This invention relates to the field of microbial production of polysaccharides. More specifically, the invention pertains to nucleic acid molecules encoding enzymes involved in biosynthesis of exopolysaccharides from Methylomonas sp.
Polysaccharides are sugar polymers that have been used widely as a thickener in food and non-food industries (Sanford et al. Pure and Appl. Chem. 56: 879-892 (1984); Sutherland, Trends Biotechnol, 16(1): 41-6 (1998)). They can be found in food products such as salad dressing, jam, frozen food, bakery products, canned food and dry food. Many other applications include suspending agents for pesticides, paints and other coating agents. They can act as flocculants, binders, film-formers, lubricants and friction reducers. Furthermore, exopolysaccharides are commonly used in oil field for oil recovery.
Traditionally, industrially useful polysaccharides have been derived from algal and plant sources. Over the past decade polysaccharides derived from microbes have been found increased usage (Sanford et al. Pure and Appl. Chem. 56: 879-892 (1984)); Sutherland, Trends Biotechnol, 16(1): 41-6 (1998)). One of the commercially well-known microbial exopolysaccharide is xanthan gum. Xanthan gum is a complex exopolysaccharide produced by a gram-negative bacterium Xanthomonas campestris pv. Campestris which is a pathogen of cruciferous plants. Xanthan consists of a xcex2-1,4-linked D-glucose backbone with trisaccharides side chains composed of mannose-(xcex2-1,4)-glucuronic acid-(xcex2-1,2)-mannose attached to alternate glucose residues in the backbone by xcex1-1,3 linkages. The polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose 1-phosphate, glucose, mannose, glucuronic acid, and mannose on polyprenol phosphate carrier (Ielpi et al., J. Bacteriol. 175:2490-2500, 1993).
One of the most characterized pathways for the production of microbial exopolysaccharides is found in Xanthomonas. For example, the biosynthetic pathway of xanthan in Xanthomonas campestris comprises five stages: (i) conversion of simple sugars to nucleotidyl derivative precursors, (ii) assembly of pentasaccharide subunits attached to the inner membrane polyprenol phosphate carrier, (iii) addition of acetyl and pyruvate groups, (iv) polymerization of pentasaccharide repeat units, and (v) secretion of polymer.
Several enzymes or proteins involved in biosynthesis of xanthan and other exopolysaccharides are well known in the art. UDP-glucose pyrophosphorylase is the enzyme that catalyzes the reaction generating UDP-glucose (UTP+glucose-1-phosphate  less than -- greater than  UDP-glucose+Ppi) (Wei et al., Biochem Biophys Res Commun. 226:607-12 (1996)). UDP-glucose is the building blocks for many exopolysaccharides containing glucose.
A cluster of gum genes are found to be required for xanthan gum synthesis in Xanthomonas campestris (Katzen et al. J. Bacteriol. 180:1607-1617 (1998); Chou, F. L., et al, Biochem. Biophys. Res. Commun. 233 (1), 265-269 (1997)). For example, GumD, the glycosyltransferase, is responsible for the transfer of the first glucose to the lipid-linked intermediates in exopolysaccharide biosynthesis in Xanthomonas campestris. GumH is the protein involved in the transfer of the mannose to the lipid-linked intermediates in exopolysaccharide synthesis in Xanthomonas campestris. 
Many other genes involved in exopolysaccharide biosynthesis have been characterized or sequenced from other organisms. The epsB gene encodes the EpsB protein that is probably involved in polymerization and/or export of EPS, has been sequenced in Ralstonia sola (Huang et al, Mol. Microbiol. 16: 977-989 (1995). The espM gene encoding EspM protein has been found in the esp gene cluster from Streptococcus thermophilus (Stingele et al, J. Bacteiol. 178: 1680-1690 (1996)). Another putative polysaccharide export protein, WZA, is identified in E. coli. (Blattner et al., Science 277: 1453-1474 (1997)). Finally, the epsV gene encodes the EpsV protein, a transferase which transfers the sugar to polysaccharide intermediates, and it has also been sequence in Streptococcus thermophilus (Bourgoin et al. Plasmid 40: 44-49 (1998); Bourgoin,F., et al., Gene 233:151-161 (1999).
In spite of the abundance of information regarding gene encoding microbial exopolysaccharides, no genes involved in this pathway have been isolated or characterized from C1 utilizing organisms, such as Methylomonas. As noted above, microbial exopolysaccharides have a variety of uses and it would be an advantage to synthesize this material from an abundance and inexpensive carbon source such as methane.
The problem to be solved therefore is to identify the genes relevant to exopolysaccharide synthesis in a C1 utilizing organism for the production of exopolysaccharides in both similar and unrelated microbes. Applicants have solved the stated problem by isolating and characterizing a complete enzymatic pathway for the synthesis of exopolysaccharide from a Methylomonas sp. 
The present invention provides an isolated nucleic acid molecule encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme, selected from the group consisting of: (a) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 2xc3x97SSC, 0.1% SDS followed by 0.1xc3x97SSC, 0.1% SDS; and (c) an isolated nucleic acid molecule that is complementary to (a) or (b).
Specifically the invention provides: 1) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 293 amino acids that has at least 58% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 2) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 473 amino acids that has at least 36% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:4, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 3) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 366 amino acids that has at least 36% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:6, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 4) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 779 amino acids that has at least 35% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:8, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 5) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 472 amino acids that has at least 23% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:10, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 6) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 272 amino acids that has at least 28% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:12, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 7) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 284 amino acids that has at least 21% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:14, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 8) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 398 amino acids that has at least 26% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:16, or a second nucleotide sequence comprising the complement of the first nucleotide sequence, and 9) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 317 amino acids that has at least 51% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:18, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
The invention also provides chimeric genes comprising the isolated nucleic acid molecule of any one of the instant sequences operably linked to suitable regulatory sequences. The invention additionally provides polypeptides encoded by the instant genes.
Similarly the invention provides a transformed host cell comprising the instant chimeric genes.
Additionally the invention provides a method of obtaining a nucleic acid molecule encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme comprising: (a) probing a genomic library with the nucleic acid molecule of the present invention; (b) identifying a DNA clone that hybridizes with the nucleic acid molecule of the present invention; and (c) sequencing the genomic fragment that comprises the clone identified in step (b), wherein the sequenced genomic fragment encodes a Methylomonas sp exopolysaccharide biosynthetic enzyme.
Alternatively the invention provides a method of obtaining a nucleic acid molecule encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme comprising: (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a); wherein the amplified insert encodes a portion of an amino acid sequence encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme.
In one embodiment the invention provides a method for the production of exopolysaccharide comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of a carbon source whereby exopolysaccharide is produced, said transformed host cell comprising a set of nucleic acid molecules encoding SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; under the control of suitable regulatory sequences.
In an alternate embodiment the invention provides a mutated nucleic acid molecule encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme having an altered biological activity produced by a method comprising the steps of:
(i) digesting a mixture of nucleotide sequences of the present invention or 5-13 with restriction endonucleases wherein said mixture comprises:
a) a native microbial gene;
b) a first population of nucleotide fragments which will hybridize to said native microbial sequence;
c) a second population of nucleotide fragments which will not hybridize to said native microbial sequence;
wherein a mixture of restriction fragments are produced;
(ii) denaturing said mixture of restriction fragments;
(iii) incubating the denatured said mixture of restriction fragments of step (ii) with a polymerase;
(iv) repeating steps (ii) and (iii) wherein a mutated microbial gene is produced encoding a protein having an altered biological activity.